Dendritic cells (DCs) are potent antigen presenting cells that play an essential role initiating and guiding immune responses toward intestinal pathogens and non-pathogens. Recent studies using microscopy techniques demonstrated that the intestine contains a population of DCs that are uniquely positioned at the interface of the host with the environment. These DCs extend dendrites into the intestinal lumen to sample and respond to stimuli and because of their physical location these DCs have great potential to influence immune responses. Despite these seminal observations, studies directly assessing this DC population are lacking. The function of these cells is largely inferred from studies of related DC populations, and from studies of in vitro derived DCs treated with epithelial cell conditioned media. Furthermore the phenotypic diversity of this DC population is almost completely unexplored. In this proposal we will define the phenotypic and functional diversity of epithelium associated dendritic cells (EA-DC) from the mouse and human small intestine. The overarching hypothesis of this proposal is that intestinal EA-DC include two subtypes of DCs with dichotomous phenotypes and functions, inflammatory DCs and tolerogenic DCs. These DCs migrate from the lamina propria (LP) to become closely associated with the epithelium in response to pathogenic or non- pathogenic signals respectively, sample lumenal stimuli, including dietary vitamin A, and subsequently migrate to mesenteric lymph nodes (MLN) to initiate vitamin A dependent mucosal adaptive immune responses. In specific aim 1 we will define the phenotypic and functional diversity of the murine and human EA-DC population. These studies will evaluate this population of cells with flow cytometry to define the EA-DC subtypes and examine these cells for the expression of toll like receptors and chemokine receptors. The morphology of these EA-DC subtypes will be evaluated with electron microscopy. The function of these DC subtypes will be investigated by evaluating the ability of these cells to imprint gut homing of lymphocytes, induce IgA class switch, and stimulate and differentiate T-lymphocytes. The physical location of the EA-DC subtypes will be evaluated in models of non-pathogenic and pathogenic infections. In specific aim 2 we will define the role of dietary vitamin A in establishing the EA-DC niche and in EA-DC function by evaluating the EA-DC subtypes for the expression of specialized molecular machinery for the uptake and storage of dietary vitamin A. We will evaluate the role of dietary source of vitamin A for mucosal specific functions of the EA-DC, and we will evaluate a role for dietary vitamin A in altering DC phenotype to occupy the EA niche. Dendritic cells are the most powerful immune cells for starting and guiding the character of immune responses, thus these cells play important roles in defending against infections and in preventing unwanted harmful immune responses. The intestine contains unique populations of dendritic cells that we know very little about. This study will increase our knowledge about how these dendritic cells work to protect us from intestinal infections and prevent unwanted harmful immune responses.